GTP-binding proteins of theRho/Rac family: regulation,effectors and functions in vivoXose R. Bustelo,* Vincent Sauzeau, and Inmaculada M. Berenjeno

SummaryRho/Rac proteins constitute a subgroup of the Rassuperfamily of GTP hydrolases. Although originallyimplicated in the control of cytoskeletal events, it iscurrently known that these GTPases coordinate diversecellular functions, including cell polarity, vesiculartrafficking, the cell cycle and transcriptomal dynamics.In this review, we will provide an overview on therecent advances in this field regarding the mechanismof regulation and signaling, and the roles in vivo ofthis important GTPase family. BioEssays 29:356–370,2007. � 2007 Wiley Periodicals, Inc.

Introduction

The isolation of rhoA,(1) the first member of the Rho/Rac family

ever identified, was achieved by Richard Axel’s group in

1985 during the search for ras-related genes in Aplysia.(1) The

subsequent use of conventional cloning techniques and the

more-recent characterization of genomes revealed that

the original gene is not alone, having numerous family

counterparts in other species including, among many others,

S. cerevisiae (7 genes), A. taliana (11 genes), C. elegans

(9 genes), D. melanogaster (9 genes) and H. sapiens

(23 genes). In humans, these twenty-three different loci can

generate at least twenty-six different proteins due to alter-

native splicing events. In accordance with their homology at

the amino acid sequence level, these proteins are classified

into six subfamilies: Rho, Rac, Cdc42, Rnd, RhoBTB and

RhoT/Miro (Fig. 1). RhoBTB and RhoT proteins are also

referred to as ‘‘atypical’’ Rho/Rac GTPases because they are

very different from the other GTPase subfamilies according to

structural, regulatory and functional criteria.

Like the majority of Ras superfamily proteins, most Rho/

Rac GTPases behave as ‘‘molecular switches’’ that fluctuate

between inactive and active states, two conformations that

depend on the binding of either GDP or GTP to the GTPases,

respectively (Fig. 2). Two types of regulatory proteins control

this cycling: GEFs and GAPs (Fig. 2). GEFs promote the

exchange of GDP for GTP molecules, thereby producing the

activation of these proteins during signal transduction. GAPs

promote the hydrolysis of the bound GTP molecules, thus

allowing the transfer of the GTPase back to the inactive state at

the end of the stimulation cycle. In the GTP-bound state, these

GTPases bind to a large collection of effector molecules that,

Centro de Investigacion del Cancer and Instituto de Biologıa Molecular

y Celular del Cancer (IBMCC), CSIC-University of Salamanca,

Salamanca, Spain.

Funding agencies: XRB’s work is supported by grants from the US

National Cancer Institute (5R01-CA73735-10), the Spanish Ministry of

Education and Science (SAF2003-00028), the Castilla-Leon Autono-

mous Government (SA053A05), and the Red Tematica de Investiga-

cion Cooperativa en Cancer (RD06/0020/0001, Spanish Ministry of

Health). VS was supported by an EMBO long term postdoctoral

fellowship. IMB was partially supported by a Spanish Ministry of

Education and Science FPU fellowship (FP2000-6489). The Centro de

Investigacion del Cancer is supported by endowments from the CSIC,

University of Salamanca, Castilla-Leon Autonomous Government, the

Red Tematica de Investigacion Cooperativa de Centros de Cancer

(C03/10, Spanish Ministry of Health), and the Foundation for Cancer

Research of Salamanca.

*Correspondence to: Xose R. Bustelo, Centro de Investigacion del

Cancer and Instituto de Biologıa Molecular y Celular del Cancer

(IBMCC). CSIC-University of Salamanca, Campus Unamuno, E-37007

Salamanca, Spain. E-mail: xbustelo@usal.es

DOI 10.1002/bies.20558

Published online in Wiley InterScience (www.interscience.wiley.com).

356 BioEssays 29.4 BioEssays 29:356–370, � 2007 Wiley Periodicals, Inc.

Abbreviations: Akt, v-akt murine thymoma viral oncogene homolog;

BAFF, B-cell activating factor; Baiap, brain-specific angiogenesis

inhibitor 1-associated protein; Bcl, B-cell chronic lymphocytic leuke-

mia/lymphoma 2 (Bcl2) like; Bcl2L, Bcl2 like; Borg, binder of rho

GTPases; Cdc42Ep, Cdc42 effector protein; C3orf10, chromosome 3

open reading frame 10; Cyfip, cytoplasmic fragile X mental retardation 1

(FMR1) interacting protein; Erk, extracellular-regulated MAP kinase;-

GAP, GTPase activating protein; GEF, Guanosine nucleotide exchange

factor; GTPase, GTP hydrolase; HSC, Hematopoietic stem cell; Hspc,

Hematopoietic stem progenitor cell; Ictm, Isoprenylcysteine carboxyl

methyltransferase; Ig, immunoglobulin; IQGAP, IQ motif containing

GTPase activating protein; Limk, LIM (Lin-11, Isl-1 and Mec-3) domain

kinase 2; NADPH, nicotinamide adenine dinucleotide phosphate; Nap,

non-catalytic region of tyrosine kinase (Nck)-associated protein; Nckap,

Nck-activated protein; Pak, p21-activated kinase; Pir, p53-inducible

mRNA; Pix, Pak-interacting exchange factor; Pkn, protein kinase N;

Rce, Ras and a factor converting enzyme; RhoGDI, Rho GDP

dissociation inhibitor; Rhpn, Rhophilin; Rock, Rho-associated, coiled-

coil containing protein kinase; Smurf, Smad ubiquitination regulatory

factor; Was, Wiskott-Aldrich syndrome protein; Wasf, Was protein

family; Wave, Was protein family verprolin-homologous protein; Wrch,

Wint-1 responsive Cdc42 homolog.

Review articles

in turn, lead to the stimulation of signaling cascades that

promote general cellular responses such as cytoskeletal

change, microtubule dynamics, vesicle trafficking, cell

polarity and cell cycle progression.(2) The plasticity of Rho/

Rac proteins both in terms of subcellular localization, regula-

tion, binding to effectors and crosstalk with other cellular

pathways has put them in a central regulatory point for a quite

large number of cellular processes. Unfortunately, the toll that

we have to pay for this is the development of diseases

when these routes become dysfunctional.(3,4) This crucial role

has led to a comprehensive study on their mechanism of

regulation, to the identification of additional elements of their

signal transduction pathways, and to the characterization

of their roles in vivo. In the present work, we will give an

overall view of the recent developments in those areas,

placing special emphasis on their regulatory and biological

properties in vivo. Given that Rho, Rac and Cdc42 are the

best-characterized Rho/Rac subfamilies, we will limit our

review to these molecules. Readers can find additional

information on other aspects of Rho/Rac biology in recent

publications.(2,5)

Regulation of Rho/Rac protein activity

In order to ensure proper signaling responses to extracellular

stimuli, cells control the activity of Rho/Rac proteins through a

number of regulatory steps. These include: (1) the control of

nucleotide binding and hydrolysis by GEFs and GAPs, a

process that has been already the object of recent reviews,(6,7)

(2) the regulation of their subcellular localization, (3) the

modulation of their protein expression levels, and (4) other

regulatory events. We summarize below the advances in the

understanding of these additional regulatory layers.

Regulation of Rho/Rac proteins by changesin the subcellular localizationIn addition to GDP/GTP exchange, most Rho/Rac proteins

require the docking onto cell membranes in order to perform

their biological functions. However, unlike other Ras super-

family proteins, this anchoring step is not achieved by default

during their biosynthesis and requires, instead, a combination

of intrinsic tethering signals and cooperative signaling

events.(8) The first and most crucial of the intrinsic tethering

signals is the progressive post-translational modification of the

Figure 1. Dendrogram showing the classification of Rho/Rac subfamily members according to structural similarity criteria. Members of

each subfamily are highlighted using the same color code and grouped by shaded areas. The first symbol used for each GTPase

corresponds to that approved by the Human Genome Organization Gene Nomenclature Committee. When appropriate, other commonly

used names are also included. The same criterium has been followed in the rest of this review article.

Review articles

BioEssays 29.4 357

Figure 2. Schematic representation of the biosynthesis (top), sequestration (middle) and regulatory (bottom) cycles of Rho/Rac proteins.

In the latter case, we have included the prototypical GDP/GTP cycle as well as other regulatory steps mediated by the action of either

effectors or other biological pathways (ubiquitination, protease cleavage, internalization). The main steps in each cycle are highlighted

using dark-gray arrows. Other less common regulatory interactions are indicated in light-gray arrows (when resulting in an activation signal)

or blunted lanes (when resulting in a downmodulation signal). The enzymes catalyzing those steps are shown in green. For the sake of

simplicity, we have not included here other post-translational events of Rho/Rac proteins that have been described in the main text such as

palmitoylation. It is also still unclear whether the insertion of the GTPase into the docking membrane is achieved when in the GDP or GTP-

bound state. The latter case is not contemplated in the scheme and would require the activation of the GTPase by GEFs, the re-association

of the GTP-bound GTPase with either RhoGDI or other carrier proteins, and the subsequent delivery of the GTPase to the target membrane.

Abbreviations used are: CAAX, an acronym derived from the combination of C¼ cysteine, A¼aliphatic amino acids and X¼Met, Ser, Ala

or Gln; Cyt, cytosol; EM, endomembranes; ER, endoplasmic reticulum; FT, farnesyl transferase; GGT, geranyl-geranyl transferase;

PM, plasma membrane; PRR BP, proline rich region binding protein. Consult main text for further details.

Review articles

358 BioEssays 29.4

so-called GTPase ‘‘CAAX box’’ (Fig. 2). The first stage of this

modification is the incorporation of either a geranyl-geranyl or,

less frequently, a farnesyl group to the cysteine residue of the

CAAX box, a process catalyzed in the cytoplasm by either

type I geranyl-geranyl or farnesyl transferases, respectively

(Fig. 2).(8,9) The attachment of the isoprenoid group to the

CAAX box promotes the translocation of the GTPases to the

endoplasmic reticulum,(8,10) where the proteolytic cleavage of

the AAX tripeptide tail ensues via the isoprenyl, CAAX-specific

protease Rce1 (Fig. 2).(8,11) After this reaction, the newly

exposed a-carboxyl group of the C-terminal cysteine residue

becomes methylesterified by the carboxyl methyltransferase

Icmt (Fig. 2).(8,12) In some cases (i.e. RhoB), Rho/Rac

proteins are further modified in the endoplasmic reticulum by

the attachment of palmitate groups on additional cysteine

residues present nearby the CAAX motif.(8) The enzyme

responsible for this step is still uncharacterized in mammals.

Recent results have shown that the incorporation of farnesyl or

geranyl-geranyl groups is a conditio sine qua non for proper

membrane anchoring and biological activity of the majority of

Ras superfamily members. Instead, the endoproteolytic and

methylation steps are only essential for the subcellular

localization and biological responses of farnesylated

GTPases.(13)

The final destination of the post-translationally-modified

GTPases depends on the computation by cells of other

ancillary signals present in the GTPase C terminus (Fig. 2). In

the case of palmitoylated GTPases, one of these additional

signals is the nature of the isoprenyl group attached to the

CAAX box. Perhaps the best example for this type of regulation

is RhoB, since this protein is localized preferentially in

endomembraneswhen geranyl-geranylated and at the plasma

membrane when farnesylated.(14) In other cases, the signal

mediating proper membrane localization is a polybasic amino

acic sequence located just upstream of the CAAX box. This is

the case of Rac subfamily proteins, where small differences in

those regions are responsible for the differential localization

of Rac1, Rac2 and RhoG in lipid rafts, endosomes, and

caveolar vesicles, respectively.(15,16) Finally, in the case of

Rac1, a proline-rich domain located near the CAAX box

contributes to the translocation of this GTPase to focal

adhesion complexes via its interaction with SH3 domain

proteins such as b-Pix, a Rac1-specific GEF that is con-

stitutively located in those subcellular regions.(17)

In addition to the presence of the above structural cues,

Rho/Rac proteins need additional upstream signals in order

to move from the cytosol to target membranes and, sub-

sequently, to remain stably anchored in those structures.

RhoGDIs play important roles in this regulatory context,

because they hide the isoprenyl groups of the GTPases, an

action that favors the sequestration of the inactive GTPases in

the cytosol or organelles (Fig. 2). This property is also

important for the removal of the GTPase from the plasma

membrane at the end of the signaling process (Fig. 2). Due to

the interaction of RhoGDIs with the GTPase switch regions,

they also impede the release of GDP from the GTPase and,

consequently, contribute to the maintenance of the GTPases

in an inactive state in non-stimulated cells (Fig. 2).(18)

The dissociation of the RhoGDI from the GTPase, an

essential requisite for the activation of GTPases by GEFs and

for their subsequent association with membranes, is regulated

at different levels during signal transduction. These regulatory

steps have been mapped out extensively in the case of Rac1.

Thus, it has been shown that integrins play an important role in

this process, because they increase the affinity of Rac for lipid

rafts, a process that in turn favors the displacement of the

geranyl-geranyl motif of the GTPase from the hydrophobic

pocket of the RhoGDI and its insertion into the phospholipid

bilayer of the target membrane (Fig. 2).(19,20) Other factors

cooperating in this dissociation step include RhoGDIs dis-

placement factors (i.e. the cytoplasmic tail of the low-affinity

nerve growth factor receptor)(21) and the decrease of the

RhoGDI affinity towards Rac1 upon phosphorylation of

RhoGDI molecules by protein kinase C(22) and Pak1,(23) a

Rac1 downstream element(24) (Fig. 2). Some Rho/Rac

GTPases also require the cooperation of additional pathways

to remain anchored to membranes once they have been

liberated from the RhoGDIs. In the case of Rac1, its residence

at the plasma membrane requires in some instances integrin-

dependent signals that block the co-internalization of the

GTPase with lipid rafts(25) (Fig. 2). Taken together, these

observations indicate that the localization of Rho/Rac

GTPases is tightly modulated in time and space by a complex

system of cell type-dependent regulatory pathways.

Transcriptional regulation and/ordifferential degradationMany Rho/Rac GTPases show cell-type-specific and/or

stimulus-dependent expression. For instance, Rac2 is mostly

restricted to hematopoietic cells.(26) Rac3 is preferentially

expressed in neurons of ganglia and the central nervous

system.(27) Moreover, RhoG and RhoB proteins have been

shown to have fluctuations during the cell cycle.(28,29) RhoB

expression undergoes further regulation by extracellular

stimuli such as UV irradiation, growth factors, cytokines and

oncogenes,(30–34) a control that is facilitated by the relatively

instability of its mRNA (t1/2¼ 20 min).(32) Finally, rhoU (also

known as wrch1) is a Wnt-regulated gene.(35) Some Rho/Rac

proteins are also controlled through degradation at specific

sites in the cell. Thus, RhoA can be degraded by the ubiquitin

ligase Smurf1 in a Rac1- and Cdc42-dependent manner

(Fig. 2). This regulation contributes to inhibit the inappropriate

formation of stress fibers in certain areas of the leading edge of

the cell during the process of cell migration.(36) Partial

proteolytic cleavage also plays regulatory roles on Rho/Rac

proteins. This is the case for Cdc42, whose proteolytic

Review articles

BioEssays 29.4 359

degradation by caspases following the activation of the Fas

death receptor contributes to the activation of Fas-dependent

apoptotic events(37) (Fig. 2).

Other regulatory eventsRho/Rac GTPases can be also regulated by additional

signaling mechanisms. Thus, RhoU has a putative autoinhi-

bitory domain at its N-terminus that can be released by the

binding of Grb2, an SH3–SH2 adaptor protein. This interaction

is mediated by the recognition of an N-terminal proline-rich

region of RhoU by one of the Grb2 SH3 domains. This

interaction does not alter GDP/GTP exchange in RhoU but,

instead, promotes a more-efficient binding of this GTPase to

the downstream serine/threonine kinase Pak1.(38) Rho/Rac

proteins can also undergo phosphorylation in specific resi-

dues, a post-translational event that may influence their

interaction with RhoGDIs,(39) stability in the membrane(39,40)

and effector functions(40) (Fig. 2).

The understanding of all these regulatory steps has allowed

the development, for the first time, of drugs that can control

the signaling output of Rho family GTPases in specific

pathological states by interfering with their GDP/GTP

exchange,(41) post-translational modification,(8) and sub-

cellular localization.(42) Given the important contribution of

Rho/Rac GTPases to the progression of some human

diseases, it is likely that these current efforts will eventually

crystallize into new therapeutic agents.

Effector molecules of Rho/Rac proteins

Once activated and translocated to their specific subcellular

locations, Rho/Rac proteins interact with downstream effector

molecules to engage specific signaling cascades.(5,24) To date,

more than 70 potential effectors have been identified for

members of the Rho/Rac family (Table 1). From a structural

point of view, it is known that these effectors use distinct

residues within the switch I and switch II regions as the major

docking/recognition sites.(5,24) This structural property has

made it possible to generate GTPase point mutants that can

bind only to a subset of effectors and engage only a limited

number of downstream effects (Fig. 3A). In some instances,

the stable association of effectors requires the participation of

additional structural cues located in the polybasic C-terminal

region, the b2 sheet and/or the helices a3, a30 and a5 of the

upstream GTPases(24) (Fig. 3A,B). In other cases, it requires

the localization of the upstream GTPase in specific sites of the

cell. For instance, the functional specificity found for Rac1 and

Rac2 in neutrophils is mainly due to their differential

subcellular localization within these hematopoietic cells.(15)

Despite the large structural diversity of Rho/Rac effectors,

we have learned a number of common regulatory themes that

take place during the activation of the downstream effectors by

Rho/Rac GTPases. Thus, the tethering of effector molecules

to membranes is part of the mechanism by which they become

activated (Fig. 4). Indeed, translocation of Pak, Pkn, citron,

Rock and other effector proteins to signaling hot spots of cells

has been shown recently.(43–47) Moreover, it has been shown

that the activation of Pak1 only occurs when active Rac1 is at

the plasma membrane but not when free in the cytosol.(19)

Other results indicate that the interaction of effectors with Rho/

Rac GTPases provokes conformational changes that

shift them from autoinhibitory conformations to fully active

structures (Fig. 4). Such regulatory mechanism has been

observed for a wide collection of both catalytic (i.e. Pak,

Rock, Pkn) and non-catalytic, adaptor-like (i.e. Diaphanous,

Was, and Baiap2) effectors.(24) These changes could be self-

sufficient for activation or, alternatively, may cooperate with

other signals to promote optimal effector activation (Fig. 4A).

For example, Pkn needs RhoA binding, lipid association and

autophosphorylation events to became fully active.(24,48)

Some downstream signaling elements are also activated by

the release of trans-inhibitory factors upon the binding of the

GTPase (Fig. 4B). This is at least the case of Wasf/Wave/Scar

proteins, which get released from an inhibitory complex

formed with Nckap1–Nap125, Cyfip2–Pir121 and C3orf10–

Hspc300 upon the binding of GTP-bound Rac1 to Nckap1 and

Cyfip2.(49) It should be noted, however, that the binding of the

activated GTPase results in the inhibition, not the stimulation,

of the bound effector (Fig. 4C). This is the case, for instance, of

the interaction of Cdc42 with Cdc42Eps (also referred to as

Borgs).(50)

The final result of the modulation of the activity of these

effectors is the generation of multibranched signals that

promote, among other responses, cytoskeletal change,

vesicle trafficking and cell cycle entry (Table 1). All these

pathways have been extensively reviewed before and will not

be re-mentioned here.(2,5) However, it should be noted that the

activation of effectors might fire back on the GTPases

themselves, thus contributing to the generation of balanced

and time-restricted signals by Rho/Rac proteins. Pak family

proteins are very active in this regulatory context, since they

can modify the activity of both RhoGDIs and Rho/Rac

GEFs(23,51) (Fig. 2). These results underscore the high level

of plasticity and large number of feed-back loops taking place

in the signal transduction pathways of these GTPases.

As in the case of the regulatory elements that mediate Rho/

Rac activation, the understanding of the mode of action of the

downstream molecules has allowed the development of

inhibitory molecules for Pak and Rock family proteins.(52–54)

One of the Rock inhibitors, fasudil (also known as HA-1077

and AT877), is already being used for the treatment of patients

with cardiovascular disorders.(55)

Genetic analysis of Rho/Rac GTPase

functions in vivo

Most of the functional observations obtained with Rho/Rac

proteins have been derived from cultured cells. While this

Review articles

360 BioEssays 29.4

Table

1.

Alis

tof

sele

cte

deff

ecto

rpro

tein

sfo

rR

hoA

,R

ac1

and

Cdc42

Effecto

raTypeofPro

tein

bUpstream

GTPasec

Main

biologicalAction

Cnksr1

Scaff

old

pro

tein

(Ksr)

RhoA

Inte

racts

with

RhoA

(Rhophili

n)

and

Ras

eff

ecto

rs(R

alG

DS

)

Rtk

n1,2

Scaff

old

pro

tein

RhoA

Inte

raction

with

PD

Zpro

tein

s,

NFkB

activation

Rhpn1,2

Scaff

old

,P

DZ

conta

inin

gpro

tein

RhoA

Cyto

ske

leta

lre

gula

tion?

Ktn

1S

caff

old

pro

tein

RhoA

,R

ac1,

Cdc42

Kin

esin

bin

din

g,

vesic

ula

rtr

affi

ckin

gth

rough

mic

rotu

bule

s

Dia

ph1,2

Scaff

old

pro

tein

(Dia

1,2

)R

hoA

,R

ac1

Cyto

ske

leta

lchange

via

pro

filin

and

Baia

p

Arfi

p2

Scaff

old

pro

tein

(Por1

)R

ac1

Cyto

ske

leta

lre

gula

tion

Pard

6A

,GS

caff

old

pro

tein

(Par6a,g)

Rac1,

Cdc42

Cell

pola

rity

.Lin

ks

GT

Pases

and

atipic

alP

KC

s

Baia

p2

Scaff

old

pro

tein

(p53

IRS)

Rac1,

Cdc42

Cyto

ske

leta

lorg

aniz

ation

via

regula

tio

nof

Wasf/

Wave

pro

tein

s

IQG

AP

1,2

RhoG

AP

and

scaff

old

pro

tein

Rac1,

Cdc42

Regula

tor

of

the

cyto

ske

leto

n,

cell-

cell

conta

cts

,and

pro

lifera

tion

Was

Scaff

old

pro

tein

(Wasp)

Rac1

Cyto

ske

leta

lre

gula

tion

via

the

Arp

2/3

com

ple

x

Nck1

Scaff

old

pro

tein

with

SH

2/S

H3

dom

ain

sR

ac1

Com

ple

xfo

rmation

with

Wasp.

Sig

naltr

ansduction

Nckap1

Scaff

old

pro

tein

(Nap125,

Nap1)

Rac1

Regula

tio

nof

the

cyto

ske

leto

nvia

Wasf

pro

tein

s

Cyfip2

Scaff

old

pro

tein

(Pir121)

Rac1

Regula

tio

nof

the

cyto

ske

leto

nvia

Wasf

pro

tein

s

Cdc42S

E1,2

Scaff

old

pro

tein

(Spec1,2

)R

ac1,

Cdc42

Modula

tion

of

GT

Pase

sig

nalin

goutp

uts

IL1R

ap1

Scaff

old

pro

tein

Rac1

Inte

rleukin

sig

nalli

ng

Hspc121

Scaff

old

pro

tein

Rac1,

RhoA

,C

dc42

Regula

tio

nof

kin

ase

cascades

and

gene

expre

ssio

n

WasL

Scaff

old

pro

tein

(N-W

asp)

Cdc42

Cyto

ske

leta

lre

gula

tion

via

de

Arp

2/3

com

ple

x

Trip10

Scaff

old

pro

tein

Cdc42

Bin

din

gof

Was

tom

icro

tubule

s

Cdc42E

P1,3

,5S

caff

old

pro

tein

(Borg

s1,3

,5)

Cdc42

Regula

tio

nof

septins

Mig

-6S

caff

old

pro

tein

Cdc42

Activ

ation

of

the

Jnk

route

Wasf1

,2S

caff

old

pro

tein

(Wave

1,2

;S

car1

,2)

Cdc42,

Rac1

Cyto

ske

leta

lre

gula

tion

via

the

Arp

2/3

com

ple

x

CopG

2C

oato

mer

pro

tein

(g2-C

op)

Cdc42

Vesic

letr

affi

ckin

g(c

lath

rin

route

)

Itpr1

Inositol1,4

,5-t

riphospha

tere

cepto

rR

hoA

Calc

ium

entr

yin

endoth

elia

lcells

Plc

G1

Phospholip

ase,

Cty

pe

(PLC

-g1)

RhoA

Pro

duction

of

second

messengers

DgkQ

Dia

cylg

lycero

lkin

asey

RhoA

Dia

cylg

lycero

ldeple

tion

PI-

5-p

5K

Lip

idkin

ase

RhoA

Modula

tion

of

phospha

tidylin

ositolbip

hospha

tele

vels

SynJ2

Poly

phosphoin

ositid

ephospha

tase

Rac1

Inhib

itio

nof

recepto

rendocyto

sis

via

the

cla

thrin

route

PIK

3R

1R

egula

tory

p85

subunit

of

PIK

3C

Rac1,

Cdc42

Regula

tio

nof

PIK

3C

activi

ty,

sig

naltr

ansduction

Pld

1P

hospholip

ase,

Dty

pe

RhoA

,R

ac,

Cdc42

Pro

duction

of

phospha

tidic

acid

and

cholin

e(s

econd

messengers

)

Plc

B2

Phospholip

ase,

Cty

pe

(PLC

-b2)

Cdc42,

Rac1

Pro

duction

of

second

messengers

Cit

Serine/t

hre

onin

ekin

ase

(Citro

n)

RhoA

Citokin

esis

Pkn1,2

Serine/t

hre

onin

ekin

ase

(Prk

)R

hoA

Vesic

lere

cyc

ling,

cell

cyc

lere

gula

tion,

Pld

1activa

tion

Rock1,2

Serine/t

hre

onin

ekin

ase

(Roka,b)

RhoA

Cyto

ske

leto

n,

cyto

kin

esis

,blo

ckage

of

cell

conta

ct

inhib

itio

n

Pak1-7

Serine/t

hre

onin

ekin

ases

Rac1,

Cdc42

Cyto

ske

leta

lorg

aniz

ation,

activation

of

kin

ase

cascades

Map3K

11

Serine/t

hre

onin

ekin

ase

(Mlk

3)

Rac1,

Cdc42

Activ

ation

of

kin

ase

cascades

Prk

cA

Serine/t

hre

onin

ekin

ase

(PK

Ca)

RhoA

,R

ac1,

Cdc42

Sig

naltr

ansduction

Cdc42bpgA

,BS

erine/t

hre

onin

ekin

ase

(MR

CKa,b)

Rac1,

Cdc42

Cyto

ske

leta

lre

gula

tion

Rps6kB

1S

erine/t

hre

onin

ekin

ase

(p70

S6K,

S6K

1)

Cdc42

Regula

tio

nof

transla

tion,

cell

cyc

le

Map3K

10

Serine/t

hre

onin

ekin

ase

(Mlk

2)

Cdc42

Activ

ation

of

kin

ase

cascades

Map3K

4S

erine/t

hre

onin

ekin

ase

(Mekk4)

Cdc42

Activ

ation

of

the

Jnk

route

Tnk2

Tyro

sin

ekin

ase

(Ack)

Cdc42

Sig

naltr

ansduction,

activa

tion

of

GE

Fs

Ncf1

,2N

AD

PH

oxid

ase

com

ple

xsubunit

Rac1,

Cdc42

Supero

xid

epro

duction

CybA

NA

DP

Hoxid

ase

com

ple

xsubunit

Rac1

Supero

xid

epro

duction

Ppp1r1

2A

Regula

tory

subunit

of

phospha

tase1

RhoA

Myo

sin

light

chain

inactivation,

cyto

ske

leta

lre

gula

tio

n

(Continued)

Review articles

BioEssays 29.4 361

approach has allowed the discovery of important aspects of

their regulation and function, it has obvious limitations. For

instance, it is rather difficult to verify whether the observations

obtained using in vitro conditions can be extrapolated to more

complex situations in which primary, non-immortalized cells

are exposed to limited amounts of stimuli or to different tissue-

and cell-type crosstalk. These approaches do not provide

faithful information regarding the level of functional overlaps

among closely related GTPases, either to verify the role of

specific GTPases in the tissues where they are actually

expressed, or to study their participation in complex physio-

logical responses. To surmount some of these problems,

different research groups have generated animal models in

which specific members of the Rho/Rac subfamily and

effectors have been disrupted using homologous recombina-

tion techniques. Below, we summarize the most-recent results

achieved in this area. As a note of caution, please be adviced

that the strict comparison of the phenotypes displayed

by different genetically modified mouse strains cannot be

interpreted in absolute terms, because the lack of effect of a

null gene may not necessarily indicate less-important roles of

its encoded protein but, rather, functional complementation

events by related proteins or parallel signal transduction

pathways. The reader can find additional information about

phenotypes of transgenic mice expressing Rho/Rac proteins

and knockout animals lacking genes for Rho/Rac GEFs in

recent review articles.(6,56,57)

Genetic analysis of members of the Rac subgroupThe inactivation of the rac1, rac2, rac3 and rhoG loci has

been already achieved in mice using standard homologous

recombination techniques or, in some cases, via the use of

either tissue-specific or inducible knockout strategies. In

addition, some Rac1 effectors belonging to the Pak and Wasf

families have also been knocked out to address their role

in vivo.

The deletion of the rac1 gene provokes embryonic lethality

caused by both gastrulation defects and apoptosis of

mesodermal cells.(58) In contrast, the inactivation of the other

Rac subfamily members gives rise to more limited defects,

which are usually found in the tissues where these GTPases

are preferentially expressed. Thus, rac2�/� mice show

hematopoietic defects (see below), rac3�/� animals present

slight motor coordination problems and enhanced learning

abilities,(59) and rhoG�/� mice have some hyperactivation of

T-cell responses to antigens and minor defects in the super-

oxide pathway of neutrophils,(60,61) the cells responsible for

inflammatory responses.

The generation of inducible, cell-type-specific knockouts

for the rac1 gene and their side-by-side comparison with both

rac2�/� and double rac1�/�; rac2�/� mutant animals has

permited a comprehensive understanding of the physiological

roles of these two GTPases and their level of signaling overlap/

Table

1.

(Continued)

Effecto

raTypeofPro

tein

bUpstream

GTPasec

Main

biologicalAction

Fm

l1Form

in-lik

em

ole

cule

Rac1

Cyto

ske

leta

lorg

aniz

ation,

cell

pola

rity

and

cyto

kin

esis

Fhod1

Form

in-

like

Rac1

Cyto

ske

leta

land

transcriptionalre

gula

tio

n

Cyfip1

Actin

bin

din

gpro

tein

Rac1

Cyto

ske

leta

lorg

aniz

ation

Fln

AA

ctin

bin

din

gpro

tein

RhoA

,R

ac1,

Cdc42

Cyto

ske

leta

lre

gula

tio

n,

actin

fila

ment

cro

sslin

kin

g

Tbc1d3

Unknow

n,

Tbc

oncopro

tein

fam

ilyC

dc42

Cyto

ske

leta

lre

gula

tio

n

TubA

1Tubulin

Rac1

Inte

gra

lcom

ponent

of

mic

rotu

bule

s

KcnA

2P

ota

siu

mC

hannelsubunit

RhoA

Pota

siu

mentr

y

Sta

t3Tra

nscriptionalfa

cto

rR

ac1,

Cdc42

Tra

nscription

Nos2A

Nitric

oxid

esynth

ase

Rac1

Nitric

oxid

epro

duction

Sm

urf

2E

3ubiq

uitin

pro

tein

ligase

2R

ac1

Sm

ad

and

RhoA

ubiq

uitin

ation,

TG

Fb

recepto

rsig

nalli

ng

aE

ffecto

rnam

es

are

giv

en

inth

econsensus

nom

encla

ture

esta

blis

hed

by

the

Hum

an

Genom

eO

rganiz

ation

and

have

been

gro

uped

accord

ing

tofu

nctions.

bIn

som

ein

sta

nces,altern

ative

nam

es

used

for

the

eff

ecto

rare

giv

en

inpare

nth

esis

.cD

ata

are

derive

dfr

om

invitro

,in

viv

o,pro

teom

icand/o

rcello

mic

techniq

ues.

An

usefu

llin

kfo

rexplo

ring

furt

her

these

inte

ractions

and

the

eff

ecto

rsofoth

er

Rho/R

ac

subfa

mily

pro

tein

sis

the

Bio

GR

ID

data

base

availa

ble

onlin

e(w

ww

.thebio

gri

d.o

rg).

Review articles

362 BioEssays 29.4

specificity (Fig. 5). In the case of HSCs, the role of Rac1 and

Rac2 has been addressed using reconstitution experiments in

sublethaly irradiated immunocompromised mice and inducible

approaches of gene inactivation. These studies revealed

important functional differences between these two family

members. Rac1 has been shown to be important for the

optimal reconstitution of the hematopoietic system, having

roles both in the engraftment and retention of HSCs in the bone

marrow. Instead, rac2�/� HSCs show normal behaviour in all

these reponses. Rac1 and Rac2 also differ in the type of

intracellular responses that they regulate in HSCs. Thus, Rac1

is essential for the entry of HSCs into the cell cycle upon

extracellular stimulation as well as for their progression

through S and G2/M phases whereas Rac2 is important for

cytoskeletal responses, adhesion, spreading and Akt-depen-

dent HSC survival(62,63) (Fig. 5).

In the case of T-cells, rac2�/� mice show no apparent

problems in the differentiation of those cells in the thymus. In

contrast, mature rac2�/� T-cells display defects in T-cell

receptor clustering, actin polymerization, generation of Ca2þ

fluxes and Erk activation upon engagement of the T-cell

receptor(64) (Fig. 5). These defects are rather marginal,

Figure 3. Structural determinants for the interaction of Rho/Rac proteins with downstream effectors.A:Scheme showing the residues of

the switch (F37 and Y40) and a30 regions involved in the selective interaction of GTP-bound Rac1 with effectors. Similar data have been

obtained with other GTPases, including RhoA, RhoG and Cdc42. B: Crystal structure of GTP-bound Cdc42 associated to the Cdc42-

binding region of Was. The reader can observe the extensive contacts established by Was with the switch I, switch II,b2 sheet anda5 helix of

the GTPase.

Review articles

BioEssays 29.4 363

probably due to the compensation exerted by the endogenous

Rac1 protein present at high levels in those cells. Rac2 is also

important for the differentiation of helper T-cells to the TH1

subtype because it regulates the p38- and NFkB-dependent

induction of interferon-g, an important mediator of this

maturation step(65) (Fig. 5). The effects of the Rac1 deficiency

in T-cells have not been addressed as yet. However, as inferred

from the results obtained by Rac1 GEFs,(57) we can predict

that this GTPase will have roles in T-cell differentiation, positive

and negative selection, stimulation of phosphatidylinositol-3

kinase/Akt and the Ras/Erk routes, and overall responses to

antigens.

Figure 4. Examples of the types of signaling outputs derived from the interaction of Rho/Rac subfamily proteins and effectors. See main

text for further details. I, inhibitor.

Review articles

364 BioEssays 29.4

In the case of B-cells, rac2-deficient animals show defects

in the B-cell compartment, displaying reduced numbers of

peripheral B-cells, peritoneal B1 cells and IgM-secreting

plasma cells. Mature rac2�/� B-cells respond poorly to

stimulation of the B-cell receptor, showing reduced levels of

Ca2þ fluxes and of cell proliferation.(66) Rac1 seems to have

only a marginal and overlapping role with Rac2 in these

cells.(67) In agreement to this, the simultaneous elimination of

rac1 and rac2 genes induces an aggravation of the rac2�/�

phenotype, leading to a developmental block of B-cell

development at very immature stages.(67) This is caused by

low survival rates derived from the improper activation of the

Akt route and the inefficient expression of two anti-apoptotic

molecules, Bcl2L1 (most commonly known as Bcl-xL) and the

BAFF receptor.(67) Instead, the single rac1 gene knockout

has no detectable effects per se in this lymphoid lineage(67)

(Fig. 5).

Unlike the case of B-cells, Rac1 and Rac2 exert non-

overlapping functions in the neutrophil lineage. rac2 null

neurophils show a severe impairment of motility, adhesion,

chemotaxis and phagocytosis as well as a drastic reduction

(&60%) in the activity of the NADPH oxidase, the enzyme

complex responsible for the generation of anti-bacterial

superoxide molecules.(68,69) Recent results have shown that

the residual level of NADPH oxidase activity found in these

animals is due to the action of RhoG and, to a minor extent, of

Rac1(61,70) (Fig. 5). rac1�/� neutrophils have milder problems,

with defects detectable only in chemokine-dependent re-

sponses.(70,71) Unlike rac2�/� neutrophils, these cells do not

show significant problems in the cytoskeleton in the absence of

chemokines with the exception of defects in the RhoA-

dependent retraction of the uropod during stochastic migra-

tion(70,72) (Fig. 5).

The role of rac genes in macrophages has only begun to be

elucidated. Available reports indicate that Rac2 is important

for superoxide production and phagocytosis to some (i.e. FcgR

stimulation, IgG-sensitized sheep red blood cells) but not

all (i.e. serum-opsonized zymosan) stimuli.(73) In addition, it

is important for the migration of these cells, as evidenced by

the lack of the accumulation of exudate macrophages during

Figure 5. Representation of the main developmental routes for hematopoietic cells and the steps that are dependent on either Rho/Rac

subfamily proteins or Rho/Rac effectors. The GTPases and/or effectors involved in those steps are highlighted in blue. The processes

impaired by the gene inactivations in each hematopoietic lineage are summarized into light-brown boxes.

Review articles

BioEssays 29.4 365

the peritoneal inflammation of rac2�/� mice(73) (Fig. 5).

Contrary to Rac2, Rac1 seems to be important for regulating

macrophage cell morphology and proper lamellipodia forma-

tion(74) (Fig. 5). However, these defects do not induce any

significant defect on the migration and chemotactic responses

of this cell type.(74)

In agreement with the high levels of expression in platelets,

Rac1 seems to be the major player of the Rac subfamily in this

cell type. Its functions include the generation of lamellipodia

upon the stimulation of platelets with ADP, the induction of

proper spreading and aggregation of platelets, and the

formation of thrombi in vivo (Fig. 5). These defects are not

very severe, because Rac1-deficient animals do not experi-

ence hemorrhages.(75)

More recently, other tissue-specific rac1 gene knockouts

have begun to shed light on its function in non-hematopoietic

tissues. Thus, it has been shown that Rac1 is important for the

formation of myelin sheaths in the central nervous system.(76)

In the case of the skin, Rac1 has been shown to be important

for the integrity of hair follicles and, as consequence, mice with

a keratinocyte-specific inactivation of the rac1 locus develop a

hairless phenotype.(77) Finally, it has been shown that Rac1

and Rac2 proteins play important roles in the dendritic cells

that present antigens to T-cells.(78) Due to this, dendritic

cells lacking expression of both Rac1 and Rac2 show defective

cytoskeletal change, migration and antigen presentation

that, as a result, preclude adequate cell contacts with T-cells.

The development of this defect requires the simultaneous

deletion of both rac1 and rac2 genes, indicating that these

two proteins exert similar and additive roles in dendritic

cells.(78)

Consistent with the important role of Pak and Wasf

family proteins in Rac1 signaling, the deletion of some of

those cytoskeletal regulators has dire consequences during

embryonic development. Thus, the elimination of the pak4

gene leads to embryonic lethality due to heart development

problems. This protein is also important for the migration,

differentiation and axogenesis of spinal cord neurons (both

motorneurons and interneurons) and for the proper folding of

the caudal region of the neural tube.(79) The disruption of the

wasf2 gene also leads to embryonic lethality at later stages

(E12.5). These embryos show growth retardation, brain

ventricle malformations and vascularization deficiencies when

compared to wild-type embryos.(80,81) As expected from the

previous functional characterization of Wasf proteins, the

analysis of wasf2�/� mouse embryonic fibroblasts (MEFs)

indicates that this cytoskeletal regulator is important for the

generation of lamellipodia, Rac1-dependent actin polymeriza-

tion, and cell migration events.(81) Despite these examples, the

disruption of other Rac effectors in mice induces milder

phenotypes. Thus, wasf1�/� adult mice develop normaly but

have reduced size and experience anxiety, sensorimotor

retardation and deficits in hippocampal-dependent learning

and memory.(82) pak5�/� mice are fully viable and display no

obvious abnormalities.(83)

Genetic analysis of members of the Rho subgroupThe phenotypes of mice lacking functional rhoB and rhoC

genes have been recently described. These two mouse strains

are fully viable and fertile. When MEFs from these animals

were studied in vitro, it was found that RhoB is important for

proper cell motility but not for adhesion or spreading.(84)

However, these latter functions became diminished when

MEFs were transformed by both E1A and ras oncogenes,

suggesting that RhoB function is probably required for

oncogenic-dependent cytoskeletal responses.(84) rhoC�/�

MEFs show only cytoskeletal defects under serum-starved

conditions.(84) In contrast to these apparently mild pheno-

types, it has been observed that rhoB and rhoC have

important, although antagonistic, roles in tumor progression.

RhoB-deficient animals are more susceptible to developing

tumors when tested in skin carcinogenesis assays, indicating

that this GTPase may have tumor-suppressor properties, at

least in the case of skin.(84) Using crosses with transgenic mice

expressing the oncogenic polyomavirus middle T-antigen, it

was observed that the absence of RhoC is not important for

tumor development.(85) Despite this, rhoC�/� tumor cells are

less metastatic than the wild-type counterparts, a phenotype

attributed to the reduced migration, lower invasiveness and

poor survival of RhoC-deficient cells.(85) Despite these

advances, more work will be required to assess the relative

contributions of RhoB and RhoC to the life and pathogenesis

of animals. An important step in that direction will be the side-

by-side comparison of these two strains using identical genetic

backgrounds and conditions. In addition, it will be interesting to

generate the double RhoB/RhoC knockout to corroborate that

their functions are not overlapping in vivo.

Although the rhoA locus has not been targeted as yet,

several of the main RhoA effectors have been inactivated by

homologous recombination. These studies have revealed that

Rock1 and Rock2 are important for eyelid closure and fusion of

the ventral body wall, because the disruption of any of those

two genes give rise to neonates with omphalocele and open

eyes.(86,87) In agreement with the described routes modulated

by Rocks, keratinocytes derived from these tissues show

defective stress fiber formation and low myosin light chain

phosphorylation upon EGF stimulation.(87) This mild pheno-

type is highly dependent of the genetic background, because

the inactivation of the rock2 gene in another mouse strain

leads to placental defects and embryonic death.(88) cit�/�

animals also develop normally but they succumb to lethal

epileptic seizures during the first postnatal month.(89) This is

due to a marked reduction in the number of GABAergic

interneurons and of both dentate gyrus and cerebellar

neurons, a phenotype caused by cytokinesis defects in

neuroblast subsets.(89) More recently, it has been shown that

Review articles

366 BioEssays 29.4

cit�/� also have defects in both the survival and cytokinesis of

spermatogenic precursors, leading to a severe impairment of

testicular function.(90) The knockout of limk2, a locus encoding

a serine/threonine kinase that is activated by Rock,(5,24) also

induces defects in spermatogenesis, although they develop

normally and show no major disturbances in the adult

period.(91) Finally, rhpn2�/� animals show no detectable

defects.(92) The relatively mild phenotype of Rock-, Cit- and

Limk2-deficient animals is somewhat surprising, given the

crucial role attributed to these three kinases in general

cytoskeletal and cytokinesis events. At least in the case of

Limk2, this mild phenotype cannot be attributed to compensa-

tion effects by other Rho/Rac effectors, because Limk2-

deficient cells show a total impairment in the phosphorylation

of the main substrate of this kinase family, the cytoskeletal

regulator cofilin.(91) An intriguing possibility derived from these

results is that, at least during embryonic development, the

migration and adhesion of cells may follow different pathways

to those described in immortalized cultured cells.

Genetic analysis of members of the Cdc42 subgroupThe knockout of the cdc42 locus leads to embryonic lethality

prior to the E6.5 stage.(93) The isolation of embryonic stem

cells from E3.5 cdc42�/� blastocysts has allowed a glimpse

of the functional relevance of Cdc42 inside cells. Under

these conditions, it has been shown that Cdc42 is essential for

the phosphatidylinositol bisphosphate-mediated polymeriza-

tion of actin and, due to this, its deletion induces a highly

disorganized cytoskeleton, round-up morphologies, and

smaller cell sizes.(94) In contrast to these results, the cell-

specific inactivation of the cdc42 locus in fibroblasts does not

induce any impairment on cytoskeletal structures or cell

migration.(94) It has been argued that this result is due to

functional redundancy with other Rho/Rac proteins, because

the expression of a dominant negative mutant of Cdc42 in

the cdc42�/� fibroblasts significantly impairs most of those

biological processes.(95) cdc42�/� cells do show defects in

polarity, including minor disturbances in establishment of the

proper directionality and relocation of the Golgi apparatus in

migrating fibroblasts.(95) These results seem to be however

highly dependent on the fibroblast type, because a more

recent study has shown that primary fibroblasts do show

problems in filopodia formation, migration and proliferation in

the absence of Cdc42 expression.(94) More recently, the

specific inactivation of the cdc42gene in oligodendrocytes and

neuronal precursors has revealed a role for Cdc42 in the

central nervous system.(76) In the case of oligodendrocytes,

Cdc42 is important for the correct formation of myelin

sheaths.(76) In the case of neuronal precursors, Cdc42

plays crucial roles in the establishment of Par6-dependent

apico-basal polarity processes of stem cells.(96) In contrast, it

does not seem important for the adhesion, cell-cycle regula-

tion or cytokinesis of this stem cell population.(96)

Several Cdc42 effectors have been also targeted by

homologous recombination. Was-deficient mice show re-

duced numbers of thymocytes, mature lymphocytes and

platelets. The reduced production of thymocytes is due to

impaired progression from the CD44�/CD25þ to the CD44�/

CD25� stage of differentiation. was�/� thymocytes and

mature T cells show impaired T-cell receptor capping and

endocytosis, generation of Ca2þ fluxes and actin polymeriza-

tion. As a consequence, they proliferate poorly upon engage-

ment of the T-cell receptor(97) (Fig. 5). Probably due to all these

immunological disturbances, was�/� mice develop colitis as

they age.(97) These animals have also neurophils with reduced

phagocytic activity and osteoclasts with severe cytoskeletal

defects that generate abnormal patterns of bone resorp-

tion.(98,99) Iqgap1�/� animals show no detectable phenotypic

defects with the exception of the development of gastric

hyperplasia,(100) a result that suggests that this Cdc42 effector

may exert inhibitory properties for the proliferation of intestinal

epithelial cells.

Taken together, these studies confirm the important role of

specific Rho/Rac family members in the biological pathways

related to cytoskeletal dynamics, polarity, cell survival/

apoptosis, cell proliferation, immune system responses and

oncogenesis. In addition, they show that despite the high

structural homology, these proteins exert related, but not

identical, functions in vivo at least in certain cell types.

Conclusion

Since the isolation of the first Rho/Rac family more than

20 years ago, substantial information has been gained

regarding the number of family members, the type of effectors

they engage, the main regulatory layers controling their

activities and the biological processes that they are implicated

on. Despite these advances, more information remains to be

gathered in the near future. For instance, we have to delineate

the dynamics and kinetics of engagement of the different

interactive Rho/Rac-dependent networks during cell signaling.

Likewise, we need to get additional information regarding the

type of signaling networks engaged and signaling outputs

generated in function of the type, concentration, and/or

combination of the extracellular stimuli received by cells.

Given the complex array of signaling molecules involved and,

in some instances, the multifunctional nature of them, the

execution of this aim will not be an easy task. Fortunately,

the high-throughput techniques that are being developed in the

cellomics field to monitor the behavior of molecules in real time

will probably help tackling these issues. Likewise, proteomic

and genomic techniques will be also useful for isolating all the

signaling complexes and regulatory molecules involved in

these pathways. Given that most of the studies done up to now

have focused on few GTPases, more work remains be done to

elucidate the functions of the less-studied family counterparts.

In this context, the generation of new animal models will help

Review articles

BioEssays 29.4 367

assigning specific functional tasks to these GTPases and, in

addition, provide information about the level of signaling

overlap and/or cooperativity existing among them. Given

the important roles that these GTPases play in different

pathologies, it is likely that the progress in these areas of

research will contribute to a better understanding and

treatment of human disease.

Acknowledgments

The authors wish to apologize to the scientists not cited in this

work due to space constrains. We also like to thank M. Dosil

and P. Crespo for helpful comments on the manuscript.

References1. Madaule P, Axel R. 1985. A novel ras-related gene family. Cell 41:31–

40.

2. Etienne-Manneville S, Hall A. 2002. Rho GTPases in cell biology. Nature

420:629–635.

3. Boettner B, Van Aelst L. 2002. The role of Rho GTPases in disease

development. Gene 286:155–174.

4. Sahai E, Marshall CJ. 2002. RHO-GTPases and cancer. Nat Rev Cancer

2:133–142.

5. Jaffe AB, Hall A. 2005. Rho GTPases: biochemistry and biology. Annu

Rev Cell Dev Biol 21:247–269.

6. Rossman KL, Der CJ, Sondek J. 2005. GEF means go: turning on RHO

GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell

Biol 6:167–180.

7. Peck J, Douglas Gt, Wu CH, Burbelo PD. 2002. Human RhoGAP

domain-containing proteins: structure, function and evolutionary rela-

tionships. FEBS Lett 528:27–34.

8. Winter-Vann AM, Casey PJ. 2005. Post-prenylation-processing en-

zymes as new targets in oncogenesis. Nat Rev Cancer 5:405–412.

9. Casey PJ, Seabra MC. 1996. Protein prenyltransferases. J Biol Chem

271:5289–5292.

10. Choy E, Chiu VK, Silletti J, Feoktistov M, Morimoto T, et al. 1999.

Endomembrane trafficking of ras: the CAAX motif targets proteins to the

ER and Golgi. Cell 98:69–80.

11. Boyartchuk VL, Ashby MN, Rine J. 1997. Modulation of Ras and a-factor

function by carboxyl-terminal proteolysis. Science 275:1796–1800.

12. Dai Q, Choy E, Chiu V, Romano J, Slivka SR, et al. 1998. Mammalian

prenylcysteine carboxyl methyltransferase is in the endoplasmic

reticulum. J Biol Chem 273:15030–15034.

13. Michaelson D, Ali W, Chiu VK, Bergo M, Silletti J, et al. 2005.

Postprenylation CAAX processing is required for proper localization of

Ras but not Rho GTPases. Mol Biol Cell 16:1606–1616.

14. Lebowitz PF, Davide JP, Prendergast GC. 1995. Evidence that

farnesyltransferase inhibitors suppress Ras transformation by interfer-

ing with Rho activity. Mol Cell Biol 15:6613–6622.

15. Filippi MD, Harris CE, Meller J, Gu Y, Zheng Y, et al. 2004. Localization

of Rac2 via the C terminus and aspartic acid 150 specifies superoxide

generation, actin polarity and chemotaxis in neutrophils. Nat Immunol

5:744–751.

16. Prieto-Sanchez RM, Bustelo XR. 2003. Structural basis for the signaling

specificity of RhoG and Rac1 GTPases. J Biol Chem 278:37916–

37925.

17. ten Klooster JP, Jaffer ZM, Chernoff J, Hordijk PL. 2006. Targeting and

activation of Rac1 are mediated by the exchange factor beta-Pix. J Cell

Biol 172:759–769.

18. DerMardirossian C, Bokoch GM. 2005. GDIs: central regulatory

molecules in Rho GTPase activation. Trends Cell Biol 15:356–363.

19. del Pozo MA, Price LS, Alderson NB, Ren XD, Schwartz MA. 2000.

Adhesion to the extracellular matrix regulates the coupling of the small

GTPase Rac to its effector PAK. Embo J 19:2008–2014.

20. Del Pozo MA, Kiosses WB, Alderson NB, Meller N, Hahn KM, et al. 2002.

Integrins regulate GTP-Rac localized effector interactions through

dissociation of Rho-GDI. Nat Cell Biol 4:232–239.

21. Yamashita T, Tohyama M. 2003. The p75 receptor acts as a

displacement factor that releases Rho from Rho-GDI. Nat Neurosci

6:461–467.

22. Price LS, Langeslag M, ten Klooster JP, Hordijk PL, Jalink K, et al. 2003.

Calcium signaling regulates translocation and activation of Rac. J Biol

Chem 278:39413–39421.

23. DerMardirossian C, Schnelzer A, Bokoch GM. 2004. Phosphorylation of

RhoGDI by Pak1 mediates dissociation of Rac GTPase. Mol Cell

15:117–127.

24. Bishop AL, Hall A. 2000. Rho GTPases and their effector proteins.

Biochem J 348Pt 2:241–255.

25. del Pozo MA, Alderson NB, Kiosses WB, Chiang HH, Anderson RG,

et al. 2004. Integrins regulate Rac targeting by internalization of

membrane domains. Science 303:839–842.

26. Didsbury J, Weber RF, Bokoch GM, Evans T, Snyderman R. 1989. rac, a

novel ras-related family of proteins that are botulinum toxin substrates. J

Biol Chem 264:16378–16382.

27. Bolis A, Corbetta S, Cioce A, de Curtis I. 2003. Differential distribution of

Rac1 and Rac3 GTPases in the developing mouse brain: implications

for a role of Rac3 in Purkinje cell differentiation. Eur J Neurosci 18:2417–

2424.

28. Zalcman G, Closson V, Linares-Cruz G, Lerebours F, Honore N, et al.

1995. Regulation of Ras-related RhoB protein expression during the cell

cycle. Oncogene 10:1935–1945.

29. Vincent S, Jeanteur P, Fort P. 1992. Growth-regulated expression of

rhoG, a new member of the ras homolog gene family. Mol Cell Biol

12:3138–3148.

30. Jahner D, Hunter T. 1991. The ras-related gene rhoB is an immediate-

early gene inducible by v-Fps, epidermal growth factor, and platelet-

derived growth factor in rat fibroblasts. Mol Cell Biol 11:3682–

3690.

31. Fritz G, Kaina B. 1997. rhoB encoding a UV-inducible Ras-related small

GTP-binding protein is regulated by GTPases of the Rho family and

independent of JNK, ERK, and p38 MAP kinase. J Biol Chem

272:30637–30644.

32. Fritz G, Kaina B, Aktories K. 1995. The ras-related small GTP-binding

protein RhoB is immediate-early inducible by DNA damaging treat-

ments. J Biol Chem 270:25172–25177.

33. Fritz G, Kaina B. 2001. Transcriptional activation of the small GTPase

gene rhoB by genotoxic stress is regulated via a CCAAT element.

Nucleic Acids Res 29:792–798.

34. Jiang K, Sun J, Cheng J, Djeu JY, Wei S, et al. 2004. Akt mediates Ras

downregulation of RhoB, a suppressor of transformation, invasion, and

metastasis. Mol Cell Biol 24:5565–5576.

35. Tao W, Pennica D, Xu L, Kalejta RF, Levine AJ. 2001. Wrch-1, a novel

member of the Rho gene family that is regulated by Wnt-1. Genes Dev

15:1796–1807.

36. Wang HR, Zhang Y, Ozdamar B, Ogunjimi AA, Alexandrova E, et al.

2003. Regulation of cell polarity and protrusion formation by targeting

RhoA for degradation. Science 302:1775–1779.

37. Tu S, Cerione RA. 2001. Cdc42 is a substrate for caspases

and influences Fas-induced apoptosis. J Biol Chem 276:19656–

19663.

38. Shutes A, Berzat AC, Cox AD, Der CJ. 2004. Atypical mechanism of

regulation of the Wrch-1 Rho family small GTPase. Curr Biol 14:2052–

2056.

39. Forget MA, Desrosiers RR, Gingras D, Beliveau R. 2002. Phosphoryla-

tion states of Cdc42 and RhoA regulate their interactions with Rho GDP

dissociation inhibitor and their extraction from biological membranes.

Biochem J 361:243–254.

40. Lang P, Gesbert F, Delespine-Carmagnat M, Stancou R, Pouchelet M,

et al. 1996. Protein kinase A phosphorylation of RhoA mediates the

morphological and functional effects of cyclic AMP in cytotoxic

lymphocytes. Embo J 15:510–519.

41. Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y. 2004. Rational design

and characterization of a Rac GTPase-specific small molecule inhibitor.

Proc Natl Acad Sci USA 101:7618–7623.

42. Pelish HE, Peterson JR, Salvarezza SB, Rodriguez-Boulan E, Chen JL,

et al. 2006. Secramine inhibits Cdc42-dependent functions in cells and

Cdc42 activation in vitro. Nat Chem Biol 2:39–46.

Review articles

368 BioEssays 29.4

43. Phee H, Abraham RT, Weiss A. 2005. Dynamic recruitment of PAK1 to

the immunological synapse is mediated by PIX independently of SLP-76

and Vav1. Nat Immunol 6:608–617.

44. Brown MC, West KA, Turner CE. 2002. Paxillin-dependent paxillin

kinase linker and p21-activated kinase localization to focal adhesions

involves a multistep activation pathway. Mol Biol Cell 13:1550–1565.

45. Dharmawardhane S, Sanders LC, Martin SS, Daniels RH, Bokoch GM.

1997. Localization of p21-activated kinase 1 (PAK1) to pinocytic

vesicles and cortical actin structures in stimulated cells. J Cell Biol

138:1265–1278.

46. Mellor H, Flynn P, Nobes CD, Hall A, Parker PJ. 1998. PRK1 is targeted

to endosomes by the small GTPase, RhoB. J Biol Chem 273:4811–

4814.

47. Kosako H, Yoshida T, Matsumura F, Ishizaki T, Narumiya S, et al. 2000.

Rho-kinase/ROCK is involved in cytokinesis through the phosphoryla-

tion of myosin light chain and not ezrin/radixin/moesin proteins at the

cleavage furrow. Oncogene 19:6059–6064.

48. Mukai H, Kitagawa M, Shibata H, Takanaga H, Mori K, et al. 1994.

Activation of PKN, a novel 120-kDa protein kinase with leucine zipper-

like sequences, by unsaturated fatty acids and by limited proteolysis.

Biochem Biophys Res Commun 204:348–356.

49. Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW. 2002.

Mechanism of regulation of WAVE1-induced actin nucleation by Rac1

and Nck. Nature 418:790–793.

50. Joberty G, Perlungher RR, Sheffield PJ, Kinoshita M, Noda M, et al.

2001. Borg proteins control septin organization and are negatively

regulated by Cdc42. Nat Cell Biol 3:861–866.

51. Callow MG, Zozulya S, Gishizky ML, Jallal B, Smeal T. 2005. PAK4

mediates morphological changes through the regulation of GEF- H1. J

Cell Sci 118:1861–1872.

52. Zhao L, Ma QL, Calon F, Harris-White ME, Yang F, et al. 2006. Role of

p21-activated kinase pathway defects in the cognitive deficits of

Alzheimer disease. Nat Neurosci 9:234–242.

53. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, et al. 1997. Calcium

sensitization of smooth muscle mediated by a Rho-associated protein

kinase in hypertension. Nature 389:990–994.

54. Nagumo H, Sasaki Y, Ono Y, Okamoto H, Seto M, et al. 2000. Rho

kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase

inhibition in smooth muscle cells. Am J Physiol Cell Physiol 278:C57–

C65.

55. Mueller BK, Mack H, Teusch N. 2005. Rho kinase, a promising drug

target for neurological disorders. Nat Rev Drug Discov 4:387–398.

56. Bustelo XR. 2002. Understanding Rho/Rac biology in T-cells using

animal models. Bioessays 24:602–612.

57. Turner M, Billadeau DD. 2002. VAV proteins as signal integrators for

multi-subunit immune-recognition receptors. Nat Rev Immunol 2:476–

486.

58. Sugihara K, Nakatsuji N, Nakamura K, Nakao K, Hashimoto R, et al.

1998. Rac1 is required for the formation of three germ layers during

gastrulation. Oncogene 17:3427–3433.

59. Corbetta S, Gualdoni S, Albertinazzi C, Paris S, Croci L, et al. 2005.

Generation and characterization of Rac3 knockout mice. Mol Cell Biol

25:5763–5776.

60. Vigorito E, Bell S, Hebeis BJ, Reynolds H, McAdam S, et al. 2004.

Immunological function in mice lacking the Rac-related GTPase RhoG.

Mol Cell Biol 24:719–729.

61. Condliffe AM, Webb LM, Ferguson GJ, Davidson K, Turner M, et al.

2006. RhoG regulates the neutrophil NADPH oxidase. J Immunol 176:

5314–5320.

62. Gu Y, Filippi MD, Cancelas JA, Siefring JE, Williams EP, et al. 2003.

Hematopoietic cell regulation by Rac1 and Rac2 guanosine tripho-

sphatases. Science 302:445–449.

63. Cancelas JA, Lee AW, Prabhakar R, Stringer KF, Zheng Y, et al. 2005.

Rac GTPases differentially integrate signals regulating hematopoietic

stem cell localization. Nat Med 11:886–891.

64. Yu H, Leitenberg D, Li B, Flavell RA. 2001. Deficiency of small GTPase

Rac2 affects T cell activation. J Exp Med 194:915–926.

65. Li B, Yu H, Zheng W, Voll R, Na S, et al. 2000. Role of the guanosine

triphosphatase Rac2 in T helper 1 cell differentiation. Science 288:

2219–2222.

66. Croker BA, Tarlinton DM, Cluse LA, Tuxen AJ, Light A, et al. 2002. The

Rac2 guanosine triphosphatase regulates B lymphocyte antigen

receptor responses and chemotaxis and is required for establishment

of B-1a and marginal zone B lymphocytes. J Immunol 168:3376–

3386.

67. Walmsley MJ, Ooi SK, Reynolds LF, Smith SH, Ruf S, et al. 2003. Critical

roles for Rac1 and Rac2 GTPases in B cell development and signaling.

Science 302:459–462.

68. Kim C, Dinauer MC. 2001. Rac2 is an essential regulator of neutrophil

nicotinamide adenine dinucleotide phosphate oxidase activation in

response to specific signaling pathways. J Immunol 166:1223–1232.

69. Li S, Yamauchi A, Marchal CC, Molitoris JK, Quilliam LA, et al. 2002.

Chemoattractant-stimulated Rac activation in wild-type and Rac2-

deficient murine neutrophils: preferential activation of Rac2 and Rac2

gene dosage effect on neutrophil functions. J Immunol 169:5043–

5051.

70. Glogauer M, Marchal CC, Zhu F, Worku A, Clausen BE, et al. 2003.

Rac1 deletion in mouse neutrophils has selective effects on neutrophil

functions. J Immunol 170:5652–5657.

71. Sun CX, Downey GP, Zhu F, Koh AL, Thang H, et al. 2004. Rac1 is the

small GTPase responsible for regulating the neutrophil chemotaxis

compass. Blood 104:3758–3765.

72. Pestonjamasp KN, Forster C, Sun C, Gardiner EM, Bohl B, et al. 2006.

Rac1 links leading edge and uropod events through Rho and myosin

activation during chemotaxis. Blood 108:2814–2820.

73. Yamauchi A, Kim C, Li S, Marchal CC, Towe J, et al. 2004. Rac2-

deficient murine macrophages have selective defects in superoxide

production and phagocytosis of opsonized particles. J Immunol

173:5971–5979.

74. Wells CM, Walmsley M, Ooi S, Tybulewicz V, Ridley AJ. 2004. Rac1-

deficient macrophages exhibit defects in cell spreading and membrane

ruffling but not migration. J Cell Sci 117:1259–1268.

75. McCarty OJ, Larson MK, Auger JM, Kalia N, Atkinson BT, et al. 2005.

Rac1 is essential for platelet lamellipodia formation and aggregate

stability under flow. J Biol Chem 280:39474–39484.

76. Thurnherr T, Benninger Y, Wu X, Chrostek A, Krause SM, et al. 2006.

Cdc42 and Rac1 signaling are both required for and act synergistically

in the correct formation of myelin sheaths in the CNS. J Neurosci

26:10110–10119.

77. Chrostek A, Wu X, Quondamatteo F, Hu R, Sanecka A, et al. 2006. Rac1

is crucial for hair follicle integrity but is not essential for maintenance of

the epidermis. Mol Cell Biol 26:6957–6970.

78. Benvenuti F, Hugues S, Walmsley M, Ruf S, Fetler L, et al. 2004.

Requirement of Rac1 and Rac2 expression by mature dendritic cells for

T cell priming. Science 305:1150–1153.

79. Qu J, Li X, Novitch BG, Zheng Y, Kohn M, et al. 2003. PAK4 kinase is

essential for embryonic viability and for proper neuronal development.

Mol Cell Biol 23:7122–7133.

80. Yamazaki D, Suetsugu S, Miki H, Kataoka Y, Nishikawa S, et al. 2003.

WAVE2 is required for directed cell migration and cardiovascular

development. Nature 424:452–456.

81. Yan C, Martinez-Quiles N, Eden S, Shibata T, Takeshima F, et al. 2003.

WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-

mediated actin-based motility. Embo J 22:3602–3612.

82. Soderling SH, Langeberg LK, Soderling JA, Davee SM, Simerly R, et al.

2003. Loss of WAVE-1 causes sensorimotor retardation and reduced

learning and memory in mice. Proc Natl Acad Sci USA 100:1723–

1728.

83. Li X, Minden A. 2003. Targeted disruption of the gene for the PAK5

kinase in mice. Mol Cell Biol 23:7134–7142.

84. Liu AX, Rane N, Liu JP, Prendergast GC. 2001. RhoB is dispensable for

mouse development, but it modifies susceptibility to tumor formation as

well as cell adhesion and growth factor signaling in transformed cells.

Mol Cell Biol 21:6906–6912.

85. Hakem A, Sanchez-Sweatman O, You-Ten A, Duncan G, Wakeham A,

et al. 2005. RhoC is dispensable for embryogenesis and tumor initiation

but essential for metastasis. Genes Dev 19:1974–1979.

86. Thumkeo D, Shimizu Y, Sakamoto S, Yamada S, Narumiya S. 2005.

ROCK-I and ROCK-II cooperatively regulate closure of eyelid and

ventral body wall in mouse embryo. Genes Cells 10:825–834.

Review articles

BioEssays 29.4 369

87. Shimizu Y, Thumkeo D, Keel J, Ishizaki T, Oshima H, et al. 2005. ROCK-

I regulates closure of the eyelids and ventral body wall by

inducing assembly of actomyosin bundles. J Cell Biol 168:941–

953.

88. Thumkeo D, Keel J, Ishizaki T, Hirose M, Nonomura K, et al. 2003.

Targeted disruption of the mouse rho-associated kinase 2 gene results

in intrauterine growth retardation and fetal death. Mol Cell Biol 23:5043–

5055.

89. Di Cunto F, Imarisio S, Hirsch E, Broccoli V, Bulfone A, et al. 2000.

Defective neurogenesis in citron kinase knockout mice by altered

cytokinesis and massive apoptosis. Neuron 28:115–127.

90. Cunto FD, Imarisio S, Camera P, Boitani C, Altruda F, et al. 2002.

Essential role of citron kinase in cytokinesis of spermatogenic

precursors. J Cell Sci 115:4819–4826.

91. Takahashi H, Koshimizu U, Miyazaki J, Nakamura T. 2002. Impaired

spermatogenic ability of testicular germ cells in mice deficient in the

LIM-kinase 2 gene. Dev Biol 241:259–272.

92. Behrends J, Clement S, Pajak B, Pohl V, Maenhaut C, et al. 2005.

Normal thyroid structure and function in rhophilin 2-deficient mice. Mol

Cell Biol 25:2846–2852.

93. Chen F, Ma L, Parrini MC, Mao X, Lopez M, et al. 2000. Cdc42 is

required for PIP(2)-induced actin polymerization and early development

but not for cell viability. Curr Biol 10:758–765.

94. Yang L, Wang L, Zheng Y. 2006. Gene Targeting of Cdc42 and

Cdc42GAP Affirms the Critical Involvement of Cdc42 in Filopodia

Induction, Directed Migration, and Proliferation in Primary Mouse

Embryonic Fibroblasts. Mol Biol Cell 17:4675–4685.

95. Czuchra A, Wu X, Meyer H, van Hengel J, Schroeder T, et al. 2005.

Cdc42 is not essential for filopodium formation, directed migration, cell

polarization, and mitosis in fibroblastoid cells. Mol Biol Cell 16:4473–

4484.

96. Cappello S, Attardo A, Wu X, Iwasato T, Itohara S, et al. 2006. The Rho-

GTPase cdc42 regulates neural progenitor fate at the apical surface.

Nat Neurosci 9:1099–1107.

97. Snapper SB, Rosen FS, Mizoguchi E, Cohen P, Khan W, et al. 1998.

Wiskott-Aldrich syndrome protein-deficient mice reveal a role for WASP

in T but not B cell activation. Immunity 9:81–91.

98. Zhang H, Schaff UY, Green CE, Chen H, Sarantos MR, et al. 2006.

Impaired integrin-dependent function in Wiskott-Aldrich syndrome

protein-deficient murine and human neutrophils. Immunity 25:285–295.

99. Calle Y, Jones GE, Jagger C, Fuller K, Blundell MP, et al. 2004. WASp

deficiency in mice results in failure to form osteoclast sealing zones and

defects in bone resorption. Blood 103:3552–3561.

100. Li S, Wang Q, Chakladar A, Bronson RT, Bernards A. 2000. Gastric

hyperplasia in mice lacking the putative Cdc42 effector IQG AP1. Mol

Cell Biol 20:697–701.

Review articles

370 BioEssays 29.4